Why shall we enrich proteins with specific
isotopes?Structural determination through NMRStructural determination through NMR
1D spectra contain structural information .. but is hard to extract
Dispersed amides: protein is folded
Downfield CH3:Protein is foldedH region
Why shall we enrich proteins with specific
isotopes?Even 2D spectra can be (and indeed are) very crowded
Realistic limit of homonuclear NMR: proteins of 100-120 amino acids; Realistic limit of homonuclear NMR: proteins of 100-120 amino acids; spectra of larger proteins are too crowdedspectra of larger proteins are too crowded
1H
1H
Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance g/107 rad T-1s-1 MHz
(2.3 T magnet) 1H 1/2 99.985 % 26.7519 100.0000002H 1 0.015 4.1066 15.35113C 1/2 1.108 6.7283 25.14514N 1 99.63 1.9338 7.22815N 1/2 0.37 -2.712 10.13678317O 5/2 0.037 -3.6279 13.56119F 1/2 100 25.181 94.09400323Na 3/2 100 7.08013 26.46631P 1/2 100 10.841 40.480737113Cd 1/2 12.26 -5.9550 22.193173
Useful nuclei such as 15N, 13C are rare
The solution is…
The solution is
3D heteronuclear NMR
Isotopic labeling
Uniform labeling
Isotopically labeled proteins can be prepared straightforwardly in E. coli by growing cells in minimal media (e.g. M9) supplemented with appropriate nutrients (15NH4Cl, 13C-glucose) or in labelled media.
Requirements for heteronuclear NMR: isotope
labeling
Residue specific labeling
Metabolic pathways can be exploited and/or appropriate auxotrophic strains of E. coli can also be used for residue selective labeling
Labeling in eukaryotic organisms
Eukaryotic proteins which are inefficiently expressed in bacteria can be efficiently expressed and labelled in yeast strains (P. pastoris)
Requirements for heteronuclear NMR: isotope
labeling Deuterium labeling
For large proteins deuterium labeling provides simplified spectra for the remaining 1H nuclei and has useful effects on relaxationproperties of attached or adjacent atoms (1H, 15N,13C).-Fractional and complete deuteration
Isotopic Uniform Labelling of proteins
15N-labelled Protein Preparation The protein is produced by expression from bacteria which are grown on minimal medium supplemented with 15NH4Cl and wild-type (wt) glucose.
15N, 13C-labelled Protein Preparation 15N,13C-labelling is commonly referred to as double labelling. The protein is produced by expression from bacteria which are grown on minimal medium supplemented with 15NH4Cl and 13C-glucose.
15N, 13C,2H-labelled Protein Preparation 15N,13C,2H-labelling is commonly referred to as triple labelling. The protein is produced by expression from bacteria which are grown on minimal medium supplemented with 15NH4Cl and 13C-glucose and using D2O instead of H2O. This will result in about 70-80% deuteration of the side-chains, as there is a certain amount of contaminating 1H present from the glucose. Higher levels of deuteration of around 95% can be achieved if 13C,2H-glucose is used. However, this is more expensive and in many cases the cheaper version is sufficient. Note that the NH groups are exchangeable. This means that they will back-exchange to 1H when the protein is purified in normal aqueous solution. In this way, many of the normal NH-based experiments can be carried out on triple-labelled protein.
The simplest labeling, and also the cheapest one is 15N, because 15N ammonia is quite cheap.
13C is more expensive, because it requires the synthesis (most commonly - biosynthesis) of 13C glucose or glycerol.
Some expression systems allow use of cheaper 13CO2.
Sources of isotopes used for uniform labeling
In most cases (except in cell-free labelling), the protein is expressed by bacteria. The isotopic labels are introduced by feeding the bacteria specific nutrients. In most cases the basis will be so-called minimal medium which contains all the salts and trace elements needed by the bacteria but contains no carbon or nitrogen sources.
These elements can then be introduced using a variety of different isotopically labelled carbon and nitrogen sources.
A standard protocol for isotope labeling
O/N Inoculum in unlabeled medium
Massive culture in labeled medium
Induction
+ IPTG
Harvesting
From protein purification to check folding
Protein isolation and purification
Protein isolation and purification will follow the standard procedure which has been set up for unlabelled protein
Protein folding can be checked by 1H NMR and 1H-15N HSQC spectra.
Check folding
How to optimize protein expression?
Choice of culture medium
Two main types of culture media can be tested for uniform labeling:
Ready-to-use media like algae or bacteria hydrolysate
Minimal media added with 15N nitrogen source or/and 13C carbon source
Minimal media
Minimal media are composed in the lab and are made of nutrients like C and N source, salts, buffering substances, traces elements and vitamins.
Carbon sourceCarbon source can be glucose (the best as gives highest yields),glycerol, acetate, succinate, methanol, Etc. In case of 13C labeling the concentration of carbon sourcecan be reduced with respect to unlabelled culture, to reduce costs!!!Checks must be performed before labelling!
Nitrogen sourceNitrogen source can be NH4Cl or (NH4) 2SO4 In case of 15N labeling the concentration of nitrogen sourcecan be reduced with respect to unlabeled culture, to reduce costs!!!Checks must be performed before labeling!
Minimal media
Minimal media are composed in the lab and are made of nutrients like C and N source, salts, buffering substances, traces elements and vitamins.
Salts Salts are NaCl/KCl, MgSO4, CaCl2
Buffer Buffer usually is phosphate, pH 7.5
Trace elements Trace elements is constituted by a mixtures of metal ions,like Co2+, Cu2+, Zn2+, Mn2+, Fe2+
Vitamins Vitamins are thiamine, biotin, folic acid, niacinamide, pantothenicacid, pyridoxal, riboflavin
Ready-to-use media
These media are usually sterile and in the correct dilutionThey can be used for massive culture in the same way as unlabeled,rich media like LB or 2 x YT.Some media yield predictable cell densities
Comparison between mineral and ready-to-use
media
Bacterial growth is usually higher in ready-to-use mediathan in minimal media.
Comparison between mineral and ready-to-use
media But protein expression?It must be tested, case by case, through expression testsExample:
Strategies to improve protein expression
An example:
Grow cell mass on unlabeled rich media allowing rapid growth to high cell density.
Exchange the cell into a labeled medium at higher cell densities optimized for maximal proteinexpression
Marley J et al. J. Biomol. NMR 2001, 20, 71-75
Strategies to improve protein expression
In practice:Cells are grown in rich unlabeled medium.When OD600 = 0.7 cells are harvested, washed withM9 salt solution, w.o. N and C source and resuspended in labeled media at a higher cell concentration.Protein expression is induced after 1 hour by addition ofIPTG.
The need of deuterationWhy is necessary to enrich the protein with Why is necessary to enrich the protein with 22H?H?
Deuteration reduces the relaxation rates of NMR-active nuclei,in particular 13C, because the gyromagnetic ratio of 2H is 6.5 times smaller than 1H
It improves the resolution and sensitivity of NMR experimentsIt improves the resolution and sensitivity of NMR experiments
Which is the ideal level of deuteration?
It depends from the size of the protein
In general
for c up to 12 ns (20 KDa) 13C/15N labeling
for c up to 18 ns (35 KDa) 13C/15N labeling and fractional deuteration
for c above 18 ns 13C/15N labeling – selective protonation and background deuteration
It depends from the type of NMR experiments
The problem to express a deuterated protein
Incorporation of 2H reduces growth rate of organisms (up to 50%) and decreases protein production as a consequence of the isotopic effect.
Deuterium labeling requires conditions different Deuterium labeling requires conditions different with respect to with respect to 1313C and C and 1515N enrichment and could N enrichment and could require bacteria adaptationrequire bacteria adaptation
Changing a hydrogen atom to deuterium represents a 100% increase in mass, whereas in replacing carbon-12 with carbon-13, the mass increases by only 8%. The rate of a reaction involving a C–H bond is typically 6–10 times faster than the corresponding C–D bond, whereas a 12C reaction is only ~1.04 times faster than the corresponding 13C reaction
Fractional deuteration
Random fractional deuteration can be obtained Random fractional deuteration can be obtained up to a up to a level of 70-75%, in a media with 85% Dlevel of 70-75%, in a media with 85% D22O with O with protonated glucose, without bacteria adaptationprotonated glucose, without bacteria adaptation
O/N culture unlabeled
Expressing culture labeled>20 h
Preinduction culturelabeled
2-6 hours OD600=0.3-1.2
As for 13C, 15N, 2H labelling all the conditions (strain, glucose conc.time of induction, etc.) must be optimized for each protein!!
Deuterium incorporation
Fractional deuteration of recombinant proteins determined using mass spectroscopy. ( ) deuteration with [2H]2O only. ( ) deuteration with [2H]2O and perdeuterated glucose.
O’Connell et al. Anal.Biochem. 1998, 265, 351-355
Perdeuteration
Perdeuteration can require a gradual adaptation of Perdeuteration can require a gradual adaptation of bacteria to increasing concentration of Dbacteria to increasing concentration of D22O. O.
Bacterial strains must be accurately selected in order Bacterial strains must be accurately selected in order to choose that which better acclimates to Dto choose that which better acclimates to D22O media.O media.
For each strain one or more colony must be selectedFor each strain one or more colony must be selectedwhich better survives in high level of Dwhich better survives in high level of D22O concetrationO concetration
A protocol for bacteria adaptation to deuterated medium
O/N Inoculum in unlabelled medium
40% D2O
Massive culture 99 % D2O
60% D2O 80% D2O 99 % D2O
Glycerol stock 40% D2O
Glycerol stock 60% D2O
Glycerol stock 80% D2O
Glycerol stock 99% D2O
Is it possible to avoid the adaptation phase?
Wüthrich lab has experimented a culture minimal medium Wüthrich lab has experimented a culture minimal medium supplemented with deuterated algal hydrolysate which allows us to supplemented with deuterated algal hydrolysate which allows us to eliminate cells pre-conditioningeliminate cells pre-conditioning.
Wüthrich K. et al J.Biomol.NMR 2004,29,
Composition of the Celtone-supplemented mediaBasic minimal medium
800 ml H2O or D2O100 ml M9 solution 2 ml 1M MgSO4 1 g NH4Cl 1 g D-glucose
Vitamin mix and trace elements 10 ml of Vitamin mix 2 ml Trace elements solution
Aminoacids supplements 1-3 g deuterate algal lysate (CELTONE) dissolved at 30 mg/mlantibiotics
M9 solution70 g Na2HPO4•7H2O
30 g KH2PO4
5 g NaClFor a 10X solution, dissolve the ingredients in 1 L of H2O.
Sterilize the solution by autoclaving and dilute it to 1X with H2O prior to use.
Is it possible to avoid the adaptation phase?
SOME RESULTSSOME RESULTS
Wüthrich K. et al J.Biomol.NMR 2004,29,
Medium composition Deuteration Advantage/disadvantagesMinimal medium on 60-92% no N-H/N-D exchange problemsGlucose + Celtone-d intermediate deuteration can be achievedin H2O
Minimal medium on 95-97% high deuterationGlucose + Celtone-din D2O
Backbone HN
Side-chians
Specific labelingLabeling of a protein can be Labeling of a protein can be easily achieved on specific easily achieved on specific residues with 2 strategies:residues with 2 strategies:
In a medium containing small amounts of glucose (13C labelled or unlabelled)/NH4Cl (15N labelled or unlabelled) and complemented with the labelled aminoacid(s). A mixture of the other unlabeled aminoacid(s) can be added to prevent any conversion of the labeledaminoacid(s)
In a complete labelled medium, containing great amount of all unlabeled aminoacids except those which are expected to be labeled.
Specific labeling: the main problem
The most important problem The most important problem encountered is the metabolic encountered is the metabolic conversions of the labeled aminoacids conversions of the labeled aminoacids which might occur during anabolism which might occur during anabolism and/or catabolism. and/or catabolism.
Use an auxotrophic strain.
Use a prototrophic strain with high concentration of aminoacids to inhibit some metabolic pathways.An example: Labeling of a protein with 13C15N Lys can be performed in unlabeled media with high level of 13C15N Lys to prevent lysine biosinthesis from aspartate conversion.
How to prevent this?How to prevent this?
Amino Acid Specific Labelling
The protein is produced by expression from bacteria which are grown on minimal medium supplemented with small amounts of 15NH4Cl and 13C-labelled glucose as well as labelled and unlabelled amino acids. The idea is that only those amino acids which are added in labelled form become labelled in the protein. Unfortunately, this may not always work as desired, since the E. coli metabolism and catabolism causes a degree of interconversion between amino acids. Thus, it is not possible to create a sample with any combination of labelled amino acids. The situation can be improved somewhat by using auxotrophic bacterial strains or incorporating enzyme inhibitors.
A cheaper way of labelling only certain amino acids, often called reverse labelling, involves expression from bacteria which are grown on minimal medium supplemented with 15NH4Cl and 13C-labelled glucose as well as unlabelled amino acids. This supresses the labelling of these amino acids and only those which have not been added unlabelled will be synthesised by the bacteria using the 13C-glucose as the carbon source. Again, a certain amount of scrambling may occur.
However, if complete control over the incorporation of amino acids is required, then cell-free methods must be used.
Specific labeling for assignment of 13C and 1H methyl from Ile, Leu,
Val Full deuteration precludes the use of Full deuteration precludes the use of NOEs for structure determination.NOEs for structure determination.
How to overcome the problem?How to overcome the problem?
Reintroduction of protons by using labeled amino acids
Reintroduction of protonsby using methyl selectivelly protonated metabolic precursors of aliphatic amino acids or the biosyntetic precursor of the aromatic rings.
The basic strategy of the SAIL approach is to prepare amino acids with the following features:
Labelling of six-membered aromatic rings by alternating 12C-2H and 13C-1H moieties
Stereo-selective replacement of one 1H in methylene groups by 2H.
Replacement of two 1H in each methyl group by 2H.
Stereo-selective modification of the prochiral methyl groups of Leu and Val such that one methyl is 12C(2H)3 and the other is 13C1H(2H)2.
The 20 protein-component SAIL amino acids are prepared based on these design concepts by chemical and enzymatic syntheses.
SAIL - Stereo-Array Isotope Labelling
SAIL - Stereo-Array Isotope Labelling
The production of SAIL proteins involves cell-free expression system. This approach indeed minimize metabolic scrambing effects and produces high incorporation rate of the added SAIL amino acid into the target protein.
Specific protonation at ring carbons of Phe, Tyr, and Trp on deuterated
proteins
NOEs involving aromatic protons are an NOEs involving aromatic protons are an important source of distance restraints in the important source of distance restraints in the structure calculation of perdeuterated proteins.structure calculation of perdeuterated proteins.
A selective reverse labeling of Phe, Tyr and Trp has been performed in perdeuterated proteins, using shikimic acid, a precursor of the aromatic rings.In this way the aromatic rings of the aminoacids are partially protonated (50%)
Rajesh S. et al. J.Biomol.NMR 2003, 27, 81-86
Specific protonation at ring carbons of Phe, Tyr, and Trp on deuterated
proteins
Specific protonation at ring carbons of Phe, Tyr, and Trp on deuterated
proteins
The aromatic rings of the aminoacids are partially protonated (40-56%).Higher level of protonation are observed in E.coli strains overexpressing a membrane bound transporter of shikimate
Complete protonation can be achieved using Complete protonation can be achieved using an auxotrophic strain defective in shikimate an auxotrophic strain defective in shikimate productionproduction
To obtain CH3 in perdeuterated protein sample:
α-Ketoacid Precursors for Biosynthetic Labeling of Methyl Sites
[1H,13C]-labeled pyruvate as the main carbon source in D2O-based minimal-media expression of proteins results in high
levels of proton incorporation in methyl positions of Ala, Ile(γ2 only), Leu, and Val in an otherwise highly deuterated protein.
An example of Site-specific labelling
A bacterial protein expression system with 13C,1H pyruvate as the sole carbon source in D2O media
Unfortunately, because the protons of the methyl group of pyruvate exchange with solvent, proteins are
produced with all four of the possible methyl isotopomers (13CH3, 13CH2D, 13CHD2, and 13CD3).
IVL - Ile, Val and Leu side-chain methyl groups
The IVL labelling scheme produces protein which is uniformly 2H,13C,15N-labelled, except for the Ile, Val and Leu side-chains which are labelled as follows:
The protein is produced by expression from bacteria which are grown on minimal medium in D2O using 13C,2H-glucose as the main carbon source and 15NH4Cl as the nitrogen source. One hour prior to induction α-ketobutyrate and
α-keto-isovalerate (labelled as shown below) are added to the growth medium and lead to the desired labelling of the Ile and the Val and Leu residues, respectively.
Use of α-ketobutyric and α-ketoisovaleric acids as biosynthetic precursors for the production of deuterated proteins with protonation
restricted to the Ileδ1 and Leuδ/Valγ positions, respectively.
SEGMENTAL LABELLING
Protein splicing is a posttranslational process in which internal segments (inteins) catalyze their own excision from the precursor proteins with consequent formation of a native peptide bond between two flanking external regions (exteins). Up to now more than three hundred inteins have been identified (see www.neb.com/neb/inteins.html) and many of them were extensively characterized . Their self-splicing properties were used to develop very convenient tools for protein engineering. There are two methods based on intein properties that have been used for segmental isotope labeling of proteins: Expressed Protein Ligation (EPL) andProtein Trans-Splicing (PTS).
METODI DI ARRICCHIMENTO ISOTOPICO
Uniform labeling All atoms of a selected element are represented by a single isotope
Partial labeling A selected element is present in a mixture of isotopic forms. It's not possible to use 15N of the amino acid to label because cell in which we express the protein have transaminase that make fast exchange of the label. Deuterium labeling could be done only for a portion of all hydrogens.
Site-specific labelling
In the site-specific labeling approach only certain residues, or particular atoms in some residues are isotopically labeled
Minimal mediaTrace ElementsIn 800 ml H2O dissolve 5 g Na2EDTA and correct to pH 7 Add the following in order, correcting to pH 7 after each:
FeCl3 (.6H20) 0.5 g (0.83 g) ZnCl2 0.05 g CuCl2 0.01 g CoCl2.6H2O 0.01 g H3BO3 0.01 g MnCl2.6H2O (.4H20) 1.6 g (1.35 g)
Make up to 1 litre, autoclave and store at 4°C.
M9-minimal media:
Per litre, adds:
7 g Na2HPO4 3 g KH2PO4 0.5 g NaCl
Then add:1 ml 1 M MgSO4 200 µl 1 M CaCl2 1 ml Thiamine (40 mg ml-1 stock) 10 ml Trace Elements
Also add, as necessary: 15 ml Glucose (20 % Stock) (gives 0.3 % final) 1 g NH4Cl
M9-Solution
